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The rhythmic locomotor behavior of flies and mice provides a phenotype for the identification of clock genes, and the underlying molecular mechanism is well studied. However, interestingly, when examining locomotor rhythm in the wild, several key laboratory-based assumptions on circadian behavior are not supported in natural conditions. The rooster crowing ‘cock-a-doodle-doo’ is a symbol of the break of dawn in many countries. Previously, we used domestic inbred roosters and showed that the timing of roosters' crowing is regulated by the circadian clock under laboratory conditions. However, it is still unknown whether the regulation of crowing by circadian clock is observed under natural conditions. Therefore, here we used red jungle fowls and first confirmed that similar crowing rhythms with domesticated chickens are observed in red jungle fowls under the laboratory conditions. Red jungle fowls show predawn crowing before light onset under 12:12 light : dim light conditions and the free-running rhythm of crowing under total dim light conditions. We next examined the crowing rhythms under semi-wild conditions. Although the crowing of red jungle fowls changed seasonally under semi-wild conditions, predawn crowing was observed before sunrise in all seasons. This evidence suggests that seasonally changed crowing of red jungle fowls is under the control of a circadian clock. more...

Estrogens play an important role in the pathobiology of breast cancer. In postmenopausal women, peripheral synthesis of estrogens from adrenal/ovarian androgens, dehydroepiandrosterone (DHEA) or androstenedione (Adione), by estrogen-metabolizing enzymes is important. Besides estrone (E1) and estradiol (E2), androgen metabolites, such as androstene-3β, 17β-diol (Aenediol) or 5α-androstane-3β, 17β-diol (Aanediol), are known to have estrogenic functions, although they have been studied much less in breast cancer. To precisely elucidate steroid metabolism in breast cancer patients and to identify the pathobiological role of estrogenic androgen metabolites, concentrations of DHEA, Adione, Aenediol, Aanediol, E1, and E2 in pairs of serum and tumor tissue from patients with primary breast cancer were measured by liquid chromatography-tandem mass spectrometry. Cell proliferation assays using Aenediol were performed for four breast cancer cell lines. Serous E2 concentration was extremely low in postmenopausal women; however, a marked increase in tumor tissue was observed in hormone receptor-positive cases. E1 concentration, in contrast, was sustained at a higher level, even in postmenopausal serum, and did not increase in tumor tissue irrespective of the hormone receptor status. Dehydroepiandrosterone was most abundant in all samples, and exhibited a similar pattern as Adione and Aenediol. 5α-Androstane-3β, 17β-diol was undetectable in most samples. Androstene-3β, 17β-diol proliferated estrogen receptor-apositive breast cancer cells in the absence of E2. The intratumoral increase of E2, but not E1, in hormone receptor-positive postmenopausal breast cancer tissue, as well as the proliferative role of Aenediol, was elucidated. more...

The rhythmic locomotor behavior of flies and mice provides a phenotype for the identification of clock genes, and the underlying molecular mechanism is well studied. However, interestingly, when examining locomotor rhythm in the wild, several key laboratory-based assumptions on circadian behavior are not supported in natural conditions. The rooster crowing 'cock-a-doodle-doo' is a symbol of the break of dawn in many countries. Previously, we used domestic inbred roosters and showed that the timing of roosters' crowing is regulated by the circadian clock under laboratory conditions. However, it is still unknown whether the regulation of crowing by circadian clock is observed under natural conditions. Therefore, here we used red jungle fowls and first confirmed that similar crowing rhythms with domesticated chickens are observed in red jungle fowls under the laboratory conditions. Red jungle fowls show predawn crowing before light onset under 12:12 light : dim light conditions and the free-running rhythm of crowing under total dim light conditions. We next examined the crowing rhythms under semi-wild conditions. Although the crowing of red jungle fowls changed seasonally under semi-wild conditions, predawn crowing was observed before sunrise in all seasons. This evidence suggests that seasonally changed crowing of red jungle fowls is under the control of a circadian clock. more...

Purpose: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti–human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells. Experimental Design: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed. Results: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC. Conclusions: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds. more...

Ever since the estrogen receptor (ER) beta was discovered in 1996, we have been trying to determine its value as a prognostic and/or predictive factor in breast cancer and its potential as a novel target for pharmacological intervention. Recent progress in cellular experiments has shown that ERbeta works as counter partner of ERalpha through inhibition of the transactivating function of ERalpha by heterodimerization, distinct regulation on several specific promoters by ERalpha or ERbeta, and ERbeta-specific regulated genes which are probably related to its anti-proliferative properties. Accumulated data from protein studies in breast cancer tissues indicate that positive expression of ERbeta appears to correlate with a favorable prognosis. Although the number of studies is small, a positive response to tamoxifen treatment is observed in both ERalpha- and ERbeta-positive populations. The significance of ERbeta2/cx, a splicing variant of ERbeta, remains controversial and needs to be analyzed in further studies. We postulate that a combined evaluation of ERbetacx with progesterone receptor may help the stratification of ERalpha-positive breast cancer. Epidemiological studies of hormone replacement therapy and isoflavone (genistein) consumption indicate the possible contribution of ERbeta-specific signaling in breast cancer prevention. A selective estrogen receptor modulator, which works as an antagonist of ERalpha and an agonist of ERbeta, may be a promising chemo-preventive treatment. more...

Enhancement of the therapeutic effect of conventional drugs is currently an active treatment strategy for breast cancer, as shown in the clinical application of trastuzumab with chemotherapeutic agents, which prolonged survival even for metastatic disease. Cyclo-oxygenase 2(COX-2)inhibitors, which are chemoprevention agents for familial polyposis coli, are now contributing to this strategy in combination with chemotherapeutic and endocrine drugs. As an endocrine application, overexpression of COX-2 contributes to increased expression of aromatase in the breast tumor. In addition, it is also known to promote rich micro-vessels within the tumor through up-regulation of prostaglandin E2(PGE2), which can induce vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)in cancer cells, and can directly modulate endothelial cell proliferation. Since both rich vasculature and accelerated estrogen synthesis are thought to contribute to unfavorable conditions for the response to endocrine therapy, inhibiting COX-2 with COX-2 inhibitors is a promising strategy to potentiate endocrine therapy. more...

Although it has been assumed that synchronized firing of gonadotropin-releasing hormone (GnRH) neurons is necessary for pulsatile GnRH secretion, there is no clear evidence for this. In the present study we simultaneously recorded spontaneous action potentials from multiple cells. Immortalized GnRH neurons (GT1-7 cells) were cultured on a multi-electrode dish (MED) and action potentials recorded by an extracellular recording method. One to two weeks after the beginning of culture, spontaneous action potentials appeared, exhibiting bursts composed of 5–10 action potentials. Burst activity was intermittent and periodic with mean burst intervals of 13.3 s. Furthermore, burst activity was recorded almost simultaneously from several micro-electrodes, suggesting that electrical activities of GT1-7 cells were synchronized with each other. Periodic bursts were completely and reversibly blocked by 1–5 µM tetrodotoxin, indicating that voltage-dependent Na+ channels are involved in their generation. γ-Aminobutyric acid (GABA) given at a 10-µM concentration shortened inter-burst intervals, whereas 10 µM bicuculline lengthened them. Finally, the gap junctional blockers n-octyl alcohol (1 mM) and carbenoxolone (100 µM) reversibly blocked periodic burst activity. The present study provides direct evidence that the electrical activity of GT1-7 cells exhibits synchronous and periodic bursts composed of action potentials. In addition, endogenous GABA is involved in GT1-7 cells in determining burst frequency. Although the precise mechanism of synchronized burst activities needs to be clarified, gap junctional communications among GT1-7 cells are at least partially involved. more...

Background: Endoxifen and 4-hydroxy tamoxifen are both active metabolites of tamoxifen and exert anti-tumor activity for ER+ breast cancer. However, only small numbers of paper reported biological profile of endoxifen. Methods: Cells were incubated with various concentration of endoxifen ( (Z)-4-Hydroxy-N-desmethyl Tamoxifen ( Results: IC50s of endoxifen for MCF-7 were 100 nM in estradiol (E2) deprivation condition and 500 nM in the presence of 1nM E2. These are significantly higher than those of 4-OH tamoxifen, which are 10 nM without E2 and 50 nM with E2, respectively. From the previous reports, plasma endoxifen concentrations in breast cancer patients were ranged from 20 to 200 nM according to CYP2D6 phenotype. Anti-proliferative index of 20 nM endoxifen for MCF-7 was 0.7 (indicating 30% reduction in proliferative activity), and 200 nM endoxifen was 0.5 in the absence of E2. These were 0.95 (20 nM) and 0.75 (200nM), respectively, when incubated with 1 nM E2. 10 nM 4-OH tamoxifen, which is mean concentration in patient plasma, showed about 0.6 anti-proliferative index with no regards to E2 condition. Without E2, endoxifen reduced ERα expression as previously reported, however, endoxifen increased ERα amount in the presence of E2 accompanied with decrease of HER-2 expression. This alternation of ERα and HER2 expression were identical to those observed using 4-OH tamoxifen. There was a weak tendency that ERβ expressing MCF-7 clones showed favorable response to endoxifen compared to parent MCF-7, but this was not significant. Conclusion: Endoxifen is active agent for MCF-7 cell, however, it requires 10-fold higher concentration compared to 4-OH tamoxifen to achieve IC 50. Difference of anti-proliferative effect between 20 nM and 200 nM of endoxifen are relatively small, and it might result in minor difference for clinical response, as expected from recent clinical studies of CYP2D6. This is the first time to show that endoxifen has opposite effect to ERα protein level depending on the presence or absence of E2. ERβ expression did not enhance activity of endoxifen in contrast to previous report. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-20. more...

Previously, we reported that serum from stage IV breast cancer patients diminished the cytotoxic activity of lymphokine activated killer cells (LAKs) (Abstract #4639 AACR in 2008). Thus, we next tested what the critical role for the inhibitory effect on LAKs was. Based on the study of protein detection in the serum using Cytokine Antibody Array (Ray Biotech), serum from stage IV patients seemed to have higher level of vascular endothelial growth factor (VEGF) as compared to early stage patients. To confirm this by more accurate way, we analyzed the serum level of VEGF by using ELISA in 24 breast cancer patients. When the serum level of VEGF was cut off by median (420 pg/ml), 11 patients with higher level of VEGF out of 12 are included in the patients whose serum inhibited the cyototoxic activity of LAKs. Therefore, we hypothesized that VEGF might be one of the key role for inhibitory effect on LAKs and neutralization of serum VEGF from the patients whose serum inhibited the cyototoxic activity of LAKs could have no inhibitory effect on LAKs. Briefly, PBMCs from a healthy volunteer were used as an effector cell for all patients’ serum incubation. After activation of the PBMCs using IL-2, LAKs were prepared and incubated with the individual patient's serum and washed. The LAKs were incubated with human breast cancer cell line MCF-7 as a target cell and LDH assay was performed to detect cytotoxicity of the LAKs. Fourteen out of 24 patients serum (serum 1) showed inhibitory effect on LAKs and we picked up these patients to test blocking study of serum VEGF. Briefly, serum from these 14 patients was incubated with neutralization antibody of VEGF (R16Y, Santa Cruz Biotechnology) (serum 2) before mix with LAKs. After incubation of serum 1 and serum 2 with LAKs, target cell cytotoxicity of LAKs from incubation with serum 1 showed 30-50% reduced level as compared to control LAKs activity. Interestingly, target cell cytotoxicity of all LAKs from incubation with serum 2 did not show such inhibitory effect (mean cell cytotoxicity of LAKs was 33.48% and 57.27%, serum 1 and serum 2, respectively, control; 55.73%). These data suggest that high level of serum VEGF in breast cancer patients might be a key role of inhibitory effect on LAKs and neutralization of the VEGF could help adoptively transferred LAKs as cancer immunotherapy in the patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5304. more...